1. Field of the Invention
The present invention relates generally to reagents and methods for attaching target molecules such as oligonucleotides (oligos) to a surface. The present invention further relates to the resultant coated surfaces themselves.
2. Technical Background
A biological array can contain a chosen collection of biomolecules, for example, probes specific for important pathogens, sequence markers, antibodies, immunoglobulins, receptor proteins, peptides, cells, and the like. For example, an array can contain a chosen collection of oligonucleotides specific for known sequence markers of genetic diseases or probes to isolate a desired protein from a biological sample. A biological array may comprise a number of individual biomolecules tethered to the surface of a substrate in a regular pattern, each one in a different area, so that the location of the biomolecule is known.
Biomolecule arrays can be synthesized directly on a substrate employing methods of solid-phase chemical synthesis in combination with site-directing mass as disclosed in U.S. Pat. No. 5,510,270, incorporated herein by reference; photolithographic techniques involving precise drop deposition using piezoelectric pumps, as disclosed in U.S. Pat. No. 5,474,796, incorporated herein by reference; or contacting a substrate with typographic pins holding droplets and using ink jet printing mechanisms to lay down an array matrix.
Commercially available substrates for heterogeneous assays capable of immobilization of biomolecules such as SuperAldehyde™ from CloneTech or 3D link™ slides from Surmodics' do not have appropriate capability for covalent attachment of biomolecules, e.g., smaller oligonucleotides of less than 500 nucleotides. The SuperAldehyde™ slide requires an additional reduction step to stabilize a covalent attachment between the slide and the biomolecule. This causes problems in some heterogeneous assays as the fluorescent signal from a label on the biomolecule may be reduced or damaged. The Surmodics' slides on the other hand require gentle contact or ink jet printing.
There are those who believe that the covalent attachment of the target to the surface allows for a better product. They further believe that generating the probe on the surface using methods of solid phase synthesis is a better process than attaching the final product to a modified surface. While this might avoid the complications of adding an anchor point (functionality added for the specific purpose of surface reaction) it produces the unavoidable consequence of any linear non-convergent synthesis which results in a low yield of the desired product. For example, a 10 step linear synthesis giving a 95% yield in each step gives a final yield of only 60%. The synthesis of a 20-mer gives a final yield of 36% and a 30-mer gives final yield of 21%. By the time a 50-mer is reached only 8% is the desired product is left. The other 92% are fragments left over during each synthesis step. An added complication is that each fragment may react in any subsequent synthetic step, which in turn generates any number of alternate sequences than the desired one. This has the ultimate problem of producing false positives during the hybridization reaction. It would be ideal if each synthesis reaction could produce the 95% yield which is not realistic since each step suffers some loss attributed to several factors which include, but are not limited to, bad reagents, wrong time and/or temperature, and contamination.
The case of the aldehyde surface that attaches to a primary amine to form the imine (Schiff Base) requires the use of a hydride reducing agent to stabilize the bond. The reason the bond is unstable is that imines are susceptible to hydrolysis giving back the amine and the aldehyde. Traditionally the hydride reducing agent is a borohydride in a less reactive form like the cyanoborohydride. The purpose of the cyano group is to reduce the reactivity of the protons which immediately forms H2 in the presence of water and consumes the reagent. A problem with using boron is that it forms stable complexes with amine functions that usually need rigorous conditions to break. Another problem with these reducing agents is that they react with many carbonyl groups of which an aldehyde is merely one example. Amides are another type of carbonyl group present in the bases thymine, cytosine and guanine which can also be attacked by hydride reagents.
The use of a non-covalent interaction as the primary means of attachment has been accomplished by generating a positively charged surface, which then interacts with the intrinsic negative charge of the phosphate backbone of DNA. Primary amines are those which are protonated in neutral water (buffered pH ˜7) and those that have a pKa of ˜10, have been shown to work well. Primary amines used as a means of attachment include, but are not limited to, polylysines, GAPS, dendrimers. These primary amines also generate single point charge when protonated. Hard/Soft Acid Base (HSAB) Theory concludes that the interaction of like species gives stronger interactions than unlike species. For example, common counter ions for the ammonium cation (NH4+) is a chloride (Cl−) hydroxide (HO−). These are all ions that have no capability to resonate/delocalize the charge. The phosphate group, found in the backbone of DNA, has a negative charge that can be delocalized through resonance and thereby make it a softer charge. Ideally then a softer charged surface should interact more favorably (stronger) than a surface with point charges. Therefore, it would be highly desirable to have a method and reagent composition for the attachment of target molecules onto a surface of a substrate that can eliminate the specific binding problems associated with covalent attachments between the substrate and biomolecule.